After we used a cellphone to take pictures of the samples that had a blue LED light shining through them, we processed these images with ImageJ software to find the amount of green light that was produced (IntDen). The results from this ImageJ analysis of the images can be seen below:

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[[Image:BME103_Group5_Assembly(1).jpg|300px|Flourimeter Assembly]]<br>After we used a cellphone to take pictures of the samples that had a blue LED light shining through them, we processed these images with ImageJ software to find the amount of green light that was produced (IntDen). The results from this ImageJ analysis of the images can be seen below:

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'''Figure 6 shows the amplification of the sequence to a greater magnitude than Figure 1 after more cycles''' <br> <br>

'''Figure 6 shows the amplification of the sequence to a greater magnitude than Figure 1 after more cycles''' <br> <br>

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''All images courtesy of OpenPCR''

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Source of images: http://learn.genetics.utah.edu/content/labs/pcr/

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| PCR: Patient 2 ID 66913, rep 3 || 2203129 || 7.703 || positive

| PCR: Patient 2 ID 66913, rep 3 || 2203129 || 7.703 || positive

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Despite Patient 1's second sample being positive for cancer, patient 1 is more than likely without cancer and this repetition is due to an error like contamination. More testing would be necessary to find out.

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Despite Patient 1's second sample being positive for cancer, patient 1 is more than likely without cancer and this repetition being positive is due to an error like contamination. More testing would be necessary to find out.

OUR TEAM

LAB 1 WRITE-UP

Initial Machine Testing

The Original DesignThe Polymerase Chain Reaction (PCR) machine, shown above, is used to replicate a large quantity of a specific strand of DNA. The PCR Machine performs this task by first splitting up the DNA. Since DNA is double stranded, and melts to become two seperate strands at a certain temperature, the PCR Machine heats the DNA to the specific temperature so that the DNA becomes to seperate strands of DNA. Then the PCR Machine uses primers, which are strands of DNA that contain a certain number of nucleotides, to adhere to the two seperate strips of DNA. Then the polymerase, which is an enzyme used to fill in the holes of DNA, completes the strand, resulting in two seperate, double strands of DNA.

Experimenting With the Connections

When the wire from the board for the LCD screen was unplugged from the main board, the LCD screen on the machine turned off and went blank.
When the white wire that connects the main board of the machine and the temperature system was unplugged, the temperature reading decreased and was not accurate.

Test Run
We administered a test run on October 25 and followed the protocol provided. Everything ran smoothly as the numbers on the Open PCR display screen matched the numbers shown on the computer.
One minor inconsistency was that the estimated time to complete the test did not match the actual time it took to complete it.

Protocols

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a process that uses DNA polymerase to synthesize a large number of copies of a target DNA sequence. PCR is dependent on short DNA fragments called primers. After the DNA has been denatured by heating and then cooled to a temperature suitable for the primers to bind to their complementary sequences, the primers bind to areas adjacent to each side of the targeted DNA sequence. Once the primers are in place, the polymerase extends them into large complimentary strands. The DNA is then denatured once again, then cooled, the primers bind to the complimentary sequence and then the polymerase extends them. Repeating this process results in an exponential amplification of the target DNA sequence.

Amplifying a patient's DNA sample using PCR can be done as follows:

Collect biological samples from patients or target group.

Combine samples with reagents primers to the sample. These primers will enable the DNA to "unzip" and duplicate the target region using the extra base pairs mixed into the solution.

Place the DNA sample and reagents into a PCR machine, and program the machine to carry out the desired sequences.

Allow the machine to cycle. Once complete, collect the amplified DNA and test.

After we used a cellphone to take pictures of the samples that had a blue LED light shining through them, we processed these images with ImageJ software to find the amount of green light that was produced (IntDen). The results from this ImageJ analysis of the images can be seen below:

Sample

Area

Mean

IntDen

RawIntDen

Negative control

16268

21.125

343656

343656

N.c. background

16268

.326

5297

5297

Positive control

16800

76.351

1282697

1282697

P.c. background

16800

.282

4732

4732

Patient 1, sample 1

25464

14.135

359937

359937

Patient 1, sample 1, background

25464

0.063

1603

1603

Patient 1, sample 2

26924

44.721

1204073

1204073

Patient 1, sample 2, background

26924

0.009

232

232

Patient 1, sample 3

16958

20.176

342142

342142

Patient 1, sample 3, background

16958

0.088

1486

1486

Patient 2, sample 1

15276

79.489

1214276

1214276

Patient 2, sample 1, background

15276

0.264

4040

4040

Patient 2, sample 2

22628

72.753

1646248

1646248

Patient 2, sample 2, background

22628

0.066

1493

1493

Patient 2, sample 3

22804

96.671

2204484

2204484

Patient 2, sample 3, background

22804

0.059

1355

1355

Water

31856

11.147

355104

355104

Water Background

31856

0.021

672

672

Calf Thymus

13580

61.625

836872

836872

Calf Thymus Background

12212

0.05

611

611

Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The sequence r17879961 represents a specific sequence where a Thymine is replaced by Cytosine due to a missense mutation on chromosome 22. It affects gene CHK2 that is linked to colorectal cancer. A primer binds to a specific sequence on the template DNA and tells Taq polymerase where to begin reading and adding nucleotides to synthesize a new strand of DNA. Primers are very specific in that they can only bind to a certain sequence. A backwards primer consists of 20 nucleotides that specifically are ACT TCT TAC ATT CGA TAC AT. The forward primer is TGT GAT CTT CTA TGT ATG CA. These primers will only bind to that specific sequence of r17879961 where the Cytosine is present and not the Thymine.

If the sequence is present, the primers will bind to both leading and lagging strands of the template DNA. Taq polymerase can then bind and begin synthesizing the strand. The test will come out positive because the DNA will synthesize to create double stranded DNA that the SYBR Green I dye will then bind to. This will cause the DNA to fluoresce and yield a positive result.

If the r1789961 SNP is not present, the primer will not bind to the DNA template. Because the primers tell Taq polymerase where to replicate, Taq polymerase will not have anywhere to bind. Replication will not occur with the product of a double-stranded DNA, but linearly. The SYBR Green I dye only binds to double-stranded DNA, so it will not show because it is single-stranded. There would not be enough double-stranded DNA to fluoresce if the cancer gene is not present. Therefore, the test will display a negative result.

Figure 1 shows the sequence of DNA containing the sequence containing the missense that leads to colon cancer.

Figure 2 shows the DNA primers specific to the sequence attaching.

Figure 3 shows Taq polymerase recognizing where to attach due to the primers.

Figure 4 shows a successful double-stranded replication of the original DNA.

Figure 5 shows the desired sequence replicated after several cycles

Figure 6 shows the amplification of the sequence to a greater magnitude than Figure 1 after more cycles

Despite Patient 1's second sample being positive for cancer, patient 1 is more than likely without cancer and this repetition being positive is due to an error like contamination. More testing would be necessary to find out.

KEY

Sample = The samples were the various distinct sources of DNA measured.

Integrated Density = Using the ImageJ software, the image was split into its various color components. This value represents the amount of 'green' light measured from the sample with the blue LED light shining through the sample and subtracted from the background value, which was 'black' in color.

DNA μg/mL = The concentration of DNA in the sample as micro-grams per milliliter.

Conclusion = Samples with a concentration above a certain threshold were deemed "positive" for the cancer, while the samples with DNA concentrations below the threshold were considered to be "negative".